Upon transmission by a vector tick, Lyme disease spirochetes can establish a local skin infection, then may disseminate further to multiple tissues. Interactions with mammalian cells and extracellular matrix (ECM) are critical for colonization of diverse sites, and many B. burgdorferi adhesins that bind to cells or extracellular matrix (ECM) have been identified. The large number of adhesins, as well as the sequence variability of some, is likely to promote chronic multisystem disease and contribute to the noted diversity of human Lyme disease. Understanding this infection is complicated by the diversity of target tissues, multifunctionality of adhesins, and complexity of colonization (e.g. bloodstream survival, vascular adherence, tissue invasion, and escape from immune surveillance). To organize the plethora of receptor-ligand interactions into a framework for understanding the multisystem nature of Lyme disease, the three collaborators have taken multi-faceted genetic, biochemical and imaging approaches to fully characterize known B. burgdorferi adhesins, to discover and validate new adhesins, and to probe the functions of the full repertoire of B. burgdorferi adhesins using in vitro and in vivo models. These approaches led to important new insights into the known adhesins DbpA, BBK32, and P66, and defined new adhesive functions for the OspF subfamily and for OspC, which was previously shown to be essential for full infectivity. We showed that DbpA and OspC variants are associated with different tissue tropisms, so strain-specific differences in the repertoire of adhesins may contribute to corresponding differences in clinical manifestations. We found that BBK32 and P66 have roles in vascular adherence and joint invasion, and the interactions they mediate with the vascular endothelium may reflect sequential activities analogous to those involved in lymphocyte transmigration. We hypothesize that B. burgdorferi utilizes a multitude of adhesins with distinct activities to negotiate successie steps in a multiphasic and multisystem infection. In addition, sequence variation of a subset of adhesins may promote colonization of distinct sites and contribute to strain-specific differences in tissue tropism and disease. To gain a detailed understanding of how the diverse host cell interactions mediated by the complex repertoire of B. burgdorferi adhesins coordinate vascular attachment, tissue invasion and long-term colonization of diverse tissues, we will use the power of B. burgdorferi genetic systems and diverse mouse infection models. We will expand and fully characterize B. burgdorferi adhesins that promote interactions with endothelium to define the adhesin-ligand interactions that are required for dissemination and long-term colonization of mouse tissues. We will also characterize the role of each of these interactions in promoting the multiple steps required to attach to the vascular endothelium, to transmigrate across this barrier to stably colonize tissue. The proposed work will organize complex adhesive pathways into functional groups that mediate discrete steps in the infectious processes that together result in systemic Lyme borreliosis and disseminated, persistent infection in the mouse.